Direct Spectrophotometric Method Research Articles

Effects of Intronic and Exonic Polymorphisms of Paraoxonase 1 (PON1) Gene on Serum PON1 Activity in a Korean Population

Paraoxonase 1 (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces lipid peroxide accumulation, and PON1 genetic polymorphisms in the coding region modulate serum PON1 activity. In this study, we investigated the association between 3 polymorphisms of PON1 located in intron 5 (17899insdelTT and 17974CT) and exon 6 (192QR) and serum PON1 activity. The genetic polymorphisms and serum activity of PON1 were analyzed in 153 healthy Koreans by using a direct sequencing assay and spectrophotometric method, respectively. A significant linkage disequilibrium (LD) was observed between all tested single nucleotide polymorphisms, with the strongest LD observed between 17899insdelTT and 192QR (D' = 0.984). The 17899insdelTT, 17974CT and 192QR genetic polymorphisms were associated with significant differences in serum paraoxonase activity. In multiple regression analyses, smoking, triglyceride level, high-density lipoprotein (HDL) level, and the 17899insdelTT and 192QR genetic polymorphisms were significant determinants of serum paraoxonase activity, while age, smoking, triglyceride level, HDL level, and the 192QR genetic polymorphism were significant determinants of serum arylesterase activity. These results suggest that although the 192QR genetic polymorphism in the coding region of PON1 is primarily associated with serum PON1 activity, the intronic polymorphisms are also involved in serum PON1 activity, and this association may be mediated by LD.

A Rapid and Direct Sensitive Spectrophotometric Method for the Determination of Molybdenum(VI) using Gallacetophenone Phenylhydrazone with Application to Alloy Steel Samples

A rapid, sensitive and fairly selective spectrophotometric method has been developed for the determination of molybdenum(VI) using gallacetophenone phenylhydrazone (GPPH) as an analytical reagent. The GPPH forms an orange red species of molybdenum(VI) at a pH range of 3.0‐4.0 and the complex is soluble in 20% ethanol. The Mo(VI)‐GPPH complex shows maximum absorbance at 375 nm with molar absorptivity and Sandellʼs sensitivity being 1.539x104 L/mol cm and 1.5x10-3 μg cm-2 respectively. The system obeys Beerʼs law in the range of 1.0‐6.0 μg/mL. The repeatability of the method was checked by finding the relative standard deviation. The composition of the Mo(VI)‐GPPH complex is established as 1:2 by slope analysis method. The instability constant of the complex calculated from Edmond and Birnbaumʼs method is 6.785x10-8 at room temperature. The interfering effect of various cations and anions has also been studied. The method has been applied for the determination of Mo(VI) in alloy steel samples.

Spectrophotometric methods for the simultaneous determination of binary mixture of metronidazole and diloxanide furoate without prior separation

Ratio subtraction and isosbestic point methods are two innovative spectrophotometric methods for determining the concentrations of metronidazole (I) and diloxanide furoate (II) in a mixture. Metronidazole was determined by direct spectrophotometric method at λ max 314.0 nm in the presence of diloxanide furoate in the range of 4–24 μg ml −1 with a mean recovery percentage of 99.83 ± 1.41. Two spectrophotometric methods were developed for the spectral resolution of diloxanide furoate when present in mixture with metronidazole without preliminary separation. The first method depends on measuring the absorbance at the isosbestic point at 277.2 nm in the range of 5–30 μg ml −1 with a mean recovery percentage of 99.96 ± 1.47 for diloxanide furoate. The second method is the ratio subtraction spectroscopic method for spectral isolation of diloxanide furoate present in the mixture which can be measured at 251.2 nm in the range of 5–30 μg ml −1 with a mean recovery percentage of 99.73 ± 1.33 for diloxanide furoate determination. The suggested procedures were validated using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical preparations. The methods retained their accuracy and precision when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the reported method.

A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates ( R = 0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.

Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC

A rapid, economical, reproducible, and simple direct spectrophotometric method was developed and validated for the assay of nitazoxanide in pharmaceutical formulations. Nitazoxanide concentration was estimated in water at 345 nm and pH 4.5. The method was suitable and validated for specificity, linearity, precision, and accuracy. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. The proposed method was successfully applied in the determination of nitazoxanide in coated tablets and in powders for oral suspension. This method was compared to a previously developed and validated method for liquid chromatography to the same drug. There was no significative difference between these methods for nitazoxanide quantitation.

Extracellular terpenoid hydrocarbon extraction and quantitation from the green microalgae Botryococcus braunii var. Showa

Mechanical fractionation and aqueous or aqueous/organic two-phase partition approaches were applied for extraction and separation of extracellular terpenoid hydrocarbons from Botryococcus braunii var. Showa. A direct spectrophotometric method was devised for the quantitation of botryococcene and associated carotenoid hydrocarbons extracted by this method. Separation of extracellular botryococcene hydrocarbons from the Botryococcus was achieved upon vortexing of the micro-colonies with glass beads, either in water followed by buoyant density equilibrium to separate hydrocarbons from biomass, or in the presence of heptane as a solvent, followed by aqueous/organic two-phase separation of the heptane-solubilized hydrocarbons (upper phase) from the biomass (lower aqueous phase). Spectral analysis of the upper heptane phase revealed the presence of two distinct compounds, one absorbing in the UV-C, attributed to botryococcene(s), the other in the blue region of the spectrum, attributed to a carotenoid. Specific extinction coefficients were developed for the absorbance of triterpenes at 190 nm (ε = 90 ± 5 mM −1 cm −1) and carotenoids at 450 nm (ε = 165 ± 5 mM −1 cm −1) in heptane. This enabled application of a direct spectrophotometric method for the quantitation of water- or heptane-extractable botryococcenes and carotenoids. B. braunii var. Showa constitutively accumulates ∼30% of the dry biomass as extractable (extracellular) botryococcenes, and ∼0.2% of the dry biomass in the form of a carotenoid. It was further demonstrated that heat-treatment of the Botryococcus biomass substantially accelerates the rate and yield of the extraction process. Advances in this work serve as foundation for a cyclic Botryococcus growth, non-toxic extraction of extracellular hydrocarbons, and return of the hydrocarbon-depleted biomass to growth conditions for further product generation.

Kinetic and Conventional Spectrophotometric Determination of Bumadizone in its Tablets via Oxidative Coupling with 3-Methyl-2-Benzothiazolinone Hydrazone

Two simple, sensitive and accurate spectrophotometric methods have been developed for the determination of bumadizone in bulk drug and its tablets. Both methods based on the oxidative coupling reaction with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and measuring the absorbance of the developed colors by direct or kinetic spectrophotometric method. Upon treatment of a mixture of the chromogenic reagent and drug with cerium (IV) ammonium sulfate (method I) or ferric chloride (method I), a red or violet color was developed immediately or after 30 minutes measurable at 557 nm for method I or II, respectively. The absorbance-concentration plots were rectilinear over the ranges of 1-10 μg/mL (r = 0.9999) for method I and 2-16 μg/mL (r = 0.9998) for method II. The detection limits were 0.15 and 0.27 μg/mL & the quantitation limits were 0.46 and 0.84 μg/mL for methods I and II, respectively. Different experimental parameters affecting the development and stability of the reactions products were studied and optimized. The proposed methods were applied successfully to the determination of bumadizone in its tablets, and the results obtained were in good agreement with those obtained using a comparison method.

Direct and simultaneous spectrophotometric determination of Ni (II) and Co (II) using diethanoldithiocarbamate as complexing agent

A direct spectrophotometric method for simultaneous determination of Co(II) and Ni(II), with diethanoldithiocarbamate (DEDC) as complexing agent, is proposed using the maximum absorption at 360 and 638 nm (Co(II)/DEDC) and 390 nm (Ni/DEDC). Adjusting the best metal/ligand ratio, supporting eletrolite, pH, and time of analysis, linear analytical curves from 1.0 10-6-4.0 10-4 for Co(II) in the presence of Ni 1.0 10-6-1.0 10-4 mol L-1 were observed. No further treatment or calculation processes have been necessary. Recoveries in different mixing ratios were of 99%. Interference of Fe(III), Cu(II), Zn(II) and Cd(II), and anions as NO3-, Cl-, ClO4-, citrate and phosphate has been evaluated. The method was applied to natural waters spiked with the cations.

Second order advantage in the determination of amaranth, sunset yellow FCF and tartrazine by UV–vis and multivariate curve resolution-alternating least squares

A direct spectrophotometric method for the determination of three artificial colors – amaranth, sunset yellow FCF and tartrazine – in beverages samples is proposed. The spectra were recorded between 359 and 600 nm. The spectra of the samples (just filtrated), pure dyes (concentrations ranged between 0.01 and 1.8 mg L −1 for amaranth, 0.08 and 4.4 mg L −1 for sunset yellow and 0.04 and 1.8 mg L −1 for tartrazine) and synthetic mixtures were disposed in a column-wise augmented data matrix. This kind of data structure, analyzed by multivariate curve resolution-alternating least squares (MCR-ALS) makes it possible to exploit the so called ‘second order advantage’. MCR-ALS algorithm was applied to the experimental data under the non-negativity and equality constraints. As a result, the concentration of each dye in the samples and their corresponding pure spectra were obtained. The results were validated using internal reference materials and no significant differences were found ( α = 5%) between the reference values and the ones obtained with the proposed method. The second order advantage made it possible to obtain unbiased results even in the presence of interferences.

A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol

An assay involving the direct and simultaneous determination of low micromolar concentrations (1–10 μM) of both zinc and cobalt ions suitable for metal content analyses of metalloproteins is described. The procedure exploits differences in the visible absorption spectra of the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) resulting from its complexation to Zn 2+ and/or Co 2+ ions and is based on the fit of experimental spectra to a linear addition of Beer–Lambert law. The method eliminates the need for separating or masking one of the metal ions prior to their quantification and could prove to be particularly useful in studies on Co 2+-substituted zinc proteins.

Direct and simultaneous spectrophotometric determination of Ni (II) and Co (II) using diethanoldithiocarbamate as complexing agent

A direct spectrophotometric method for simultaneous determination of Co(II) and Ni(II), with diethanoldithiocarbamate (DEDC) as complexing agent, is proposed using the maximum absorption at 360 and 638 nm (Co(II)/DEDC) and 390 nm (Ni/DEDC). Adjusting the best metal/ligand ratio, supporting eletrolite, pH, and time of analysis, linear analytical curves from 1.0 10-6-4.0 10-4 for Co(II) in the presence of Ni 1.0 10-6-1.0 10-4 mol L-1 were observed. No further treatment or calculation processes have been necessary. Recoveries in different mixing ratios were of 99%. Interference of Fe(III), Cu(II), Zn(II) and Cd(II), and anions as NO3-, Cl-, ClO4-, citrate and phosphate has been evaluated. The method was applied to natural waters spiked with the cations.

New, simple, and validated UV-spectrophotometric method for the estimation of some beta blockers in bulk and formulations

A sensitive, extraction, derivatization, evaporation and complexation-free, direct spectrophotometric method is developed for the determination of some anthypertensive drugs such as acebutolol hydrochloride (ACH), atenolol (ATE), and propranolol hydrochloride (PRH) in bulk and pharmaceutical formulations. The optimum conditions for the analysis of aqua solutions of drugs are established. The method permits the determination of ACH, ATE, and PRH over a concentration range of 37.3–111.9, 53.3–213.1 and 14.8–51.8 μg/mL, respectively. Detection and quantification limits are calculated. The obtained results showed good recoveries of 99.60, 99.20 and 99.80% with relative standard deviations of 0.82, 0.79 and 1.70% for ACH, ATE, and PRH, respectively. The repeatability and reproducibility of the drugs for aqua media are determined. Precision and accuracy of the developed methods are used for the recovery studies. The proposed method is applicable for the assay of the three drugs under investigation in dosage forms and the results are in good agreement with those obtained by the literature method.

Spectrophotometric Determination of Sparfloxacin in Pharmaceutical Preparations by Ternary Complex Formation with Pd(II) and Eosin

A simple, sensitive, and direct spectrophotometric method has been developed for the assay of sparfloxacin in bulk and pharmaceutical preparations. The proposed method is based on the formation of ternary complex between an investigated drug, palladium(II) ion and eosin in the presence of methylcellulose as surfactant and acetate buffer of pH 4.2. Spectrophotometrically, under the optimum conditions, the ternary complex showed absorption maximum at 550 nm, with apparent molar absorptivity of 2.69×104 l mol−1 cm−1, Sandell's sensitivity of 0.01458 µg ml−1 and linearity in the concentration range 1.6–16 µg ml−1. The composition of the ternary complex was studied by Job's method of continuous variation and the result indicated that the molar ratio of SPFX: Pd: eosin is 1∶1∶1. The optimum reaction conditions and other analytical parameters are evaluated. The proposed method was successfully applied for the determination of SPFX in its pharmaceutical product with mean percentage recoveries of 99.71%. The observed data has been subjected to statistical analysis, which revealed high accuracy and precision.

Direct spectrophotometric method to determing serum copper with a new water soluble reagent

目的 建立一种简便、灵敏、稳定的直接光度法测定血清铜.方法 在非离子型表面活性剂聚氧乙烯失水山梨醇油酸单脂(Tween-80)及聚氧乙烯异辛基苯基醚(Triton X-100)存在下,用水溶性试剂2-(5-硝基-2-吡啶偶氮)-5-[N-正丙基-N-(3-磺酸丙基)氨基]苯酚二钠(Nitro-PAPS)作显色剂直接光度法测定血清铜.结果 该方法测定血清铜,显色络合物最大吸收波长为570 nm,线性范围达63.0μmol/L,表观摩尔吸光系数为7.95×104L/(mol·cm).回收率为98.6%～103.1%,批内CV为2.1%～3.3%,批间CV为2.7%～3.8%,与原子吸收分光光度法比较相关良好,Y=1.01X-0.27,r=0.998 2,P＞0.05,68名健康成年人血清铜含量为9.7～24.1 μmol/L((x)±2s).结论 该法血清用量少,不必去蛋白,具有操作快速、简便、结果灵敏可靠等优点,适用于血清铜的手工测定和自动分析。

Simpler spectrophotometric assay of paracetamol in tablets and urine samples

A very fast, economical and simpler direct spectrophotometric method was investigated for paracetamol (PC) determination in aqueous medium without using any chemical reagents. The method is based on the photo-absorption of the analyte at 243 nm after dissolution in water. The change in structure of PC after addition of water was studied by comparing the corresponding FTIR spectra. Optimization studies were conducted by using a 5 μg ml −1 standard solution of the analyte. Various parameters studied include, time for stability and measurement of spectra, effect of HCl, NaOH, CH 3COOH and NH 3 for change in absorbance and shift in spectra, interference by some analgesic drugs and some polar solvents and temperature effect. After optimization, Beer's law was obeyed in the range of 0.3–20 μg ml −1 PC solution with a correlation coefficient of 0.9999 and detection limit of 0.1 μg ml −1. The newly developed method was successfully applied for PC determination in some locally available tablets and urine samples. The proposed method is very useful for quick analysis of various types of solid and liquid samples containing PC.

New simple spectrophotometric assay of total carotenes in margarines

Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of time needed to complete the analysis. The resulting analytical procedure is characterized in details in terms of the figures of merit. The method is sensitive, precise and accurate; for both, standard additions and calibration in soybean oil, recovery ranges between 98 and 102%. For the most accurate analyses the approach of standard additions is preferred but for quick routine analyses this latter can be replaced by the calibration in soybean oil. Limit of detection value (LOD = 3S.D. B/ a, where S.D. B is the standard deviation of the blank, “ a” is the slope of calibration line) as low as 12 μg TC/100 g was achieved in soybean oil enabling the sensitive detection. Concentration of TC in margarines declared as being coloured with β-carotene (carotene) ranges between 0.3 and 0.9 mg/100 g while in carrot extract-coloured margarine TC is 0.2 mg/100 g.

Evaluation of a point-of-care direct spectrophotometric method for measurement of total serum bilirubin in term and near-term neonates

To evaluate point-of-care (POC) measurement of total serum bilirubin (TSB) in the management of neonatal jaundice. TSB was measured by a POC direct spectrophotometric bilirubin method (Unistat (U/TSB)) and a standard diazo clinical laboratory method (Olympus AU640E analyzer (diazo/TSB)). Agreement between U/TSB and diazo/TSB was assessed by correlation coefficient and Bland-Altman analysis. Transcutaneous bilirubin (TcB) was measured using JM-103 (JM). Correlation between U/TSB and diazo/TSB was 0.99 (n = 120). Maximum difference (U/TSB minus diazo/TSB) was -2.9 mg/dl, and 79% were +/-1 mg/dl; the average difference was -0.37+/-0.70 mg/dl and the average absolute difference was 0.60+/-0.52 mg/dl. Median time to determine U/TSB was 5 min. Correlation between U/TSB and JM was 0.92 (n = 113). Maximum difference (U/TSB minus JM) was 6.3 mg/dl, and 45% were +/-1 mg/dl; the average difference was 0.7+/-1.8 mg/dl and the average absolute difference was 1.4+/-1.2 mg/dl. Measurement of TSB using Unistat provides excellent agreement with diazo/TSB and rapid turnaround time. This technique may provide reliable POC confirmation of TcB results that are above a screening cutoff value.

Direct and derivative spectrophotometric method for the determination of nickel(II) with 2,4-dihydroxy benzaldehyde isonicotinoyl hydrazone

A simple and sensitive spectrophotometric method has been developed for the determination of nickel(II) in aqueous medium. The metal ion forms an yellow coloured soluble complex with 2,4-dihydroxy benzaldehyde isonicotinoyl hydrazone (2,4-DHBINH) at pH 6.0 with λ max at 400 nm. Beer's law is obeyed in the range of 0.059-1.17 μg ml -1 of Ni II . The molar absorptivity and the Sandell's sensitivity of the method are 4.00 x 10 4 L mol -1 cm -1 and 0.0015 μg cm -2 respectively. The interference of various diverse ions has been studied. The complex has a 1: 1 [Ni11: 2,4-DHBINH] stoichiometry. A method for the determination of nickel(II) by first order derivative spectrophotometry has also been proposed.

Autoadaptative sequential injection system for nitrite determination in wastewaters

A novel autoadaptative sequential injection system for the analysis of nitrite is described. The automatic determination uses a direct spectrophotometric method, based on the Griess–Ilosvay reaction. In this method the absorbance of the purple azo dye formed is measured at 555 nm. In the sequential injection operation, the sample and the reagent are aspirated and mixed by reverse flow. The sequencing and overlapping of stacked (reagent) zones as well as selection of volumes have been studied in detail. The proposed analytical system is intelligent, simple and robust, allowing for nitrite determination in a double concentration range, by a simple and automatic programmable operation change. These two ranges are 0.0–3.0 and 0.0–20.0 ppm with detection limits being 0.048 and 0.4 ppm, respectively. Next surroundings have been developed allowing autocalibration and independent monitoring of nitrite concentration. The experimental set-up has been evaluated applying it to real samples analysis of very diverse concentration samples coming from a WWTP. The throughput of the method was 12 samples per hour.

A very sensitive flow system for the direct determination of copper in natural waters based on spectrophotometric detection

A very sensitive flow injection method with spectrophotometric detection has been developed for the on-line determination of copper in natural waters. The method exhibits a limit of detection three times lower than the most sensitive direct spectrophotometric method previously described and then allows the direct and simple in situ determination of copper in most natural waters. The method was based on the measurement of the absorbance of the coloured complex formed by copper with the chromogenic reagent di-2-pyridyl ketone benzoylhydrazone (dPKBH) in an alkaline medium. This complex presents stoichiometry 1:2 (Cu:dPKBH), and exhibits maximum absorbance at 370 nm. The manifold used was very simple, and consisted of two channels. The first one contained the sample while the second one contained the colorimetric reagent (3.3×10 −4 M dPKBH in 10% ethanol), in a 1.6×10 −2 M phosphate buffer solution at pH 8. The performance of the system was optimised by using both univariate and modified simplex methodologies. When modified simplex was used, the best signal was obtained for a sample injection volume of 529 μl, a reaction coil length of 1.29 m, and a reagent flow rate of 4.8 ml min −1. Under optimum conditions, the response was linear up to 3 mg l −1 copper, the equation of the straight line being y=0.314 x+5.2×10 −4 ( r 2=0.998). The method allowed a sampling frequency of 40 samples per hour and exhibited a precision of 2.11% (as R.S.D., n=11). The limit of detection was 4.6 μg l −1 (calculated as 3 s b/ m, where s b is the standard deviation of the y-intercept and m represents the slope of the straight line), and was therefore more sensitive than all the direct continuous methods reported previously. The method was successfully applied to the analysis of real water samples, with an average relative error of 5.32%.